The present invention relates to processing biological molecules, particularly to imaging biological molecules with an atomic force microscope (AFM), and more particularly to a method and apparatus for imaging, cutting, and collecting biological molecules with nanometer resolution, and measuring hardness of materials.
An atomic force microscope (AFM) scans over the surface of a sample in two different modes of operation. In one mode, known as the contacting mode, a sharp tip is mounted on the end of a cantilever and the tip rides on the surface of a sample with an extremely light tracking force, on the order of 10.sup.-5 to 10.sup.-10 Newtons (N). In the contacting mode of operation, profiles of the surface topology are obtained with extremely high resolutions. Images showing the position of individual atoms are routinely obtained. In the other mode, the tip is held a short distance, on the order of 5 to 500 Angstroms, from the surface of a sample and is deflected by various forces between the sample and the tip, such forces include electro-static, magnetic, and van der Waals forces.
Several methods of detecting the deflection of the cantilever are available which have sub-angstrom sensitivity, including vacuum tunneling, optical interferometry, optical beam deflection, and capacitive techniques. However, fabrication of a readily reproducible cantilever stylus assembly has been a limiting factor on use of AFM and other forms of microscopy, such as scanning tunneling microscopes.
A typical cantilever stylus assembly includes a cantilever arm and a protruding tip on the arm, and in certain applications it is desirable that the cantilever flex in only one direction and have high lateral stiffness. Also, it is often required that a conductive electrode be located on the cantilever opposite the tip. In addition, the protruding tip must be sharp, that is with a radius less than 500 Angstroms and which may terminate in a single atom to provide good lateral resolution.
Substantial effort has gone into developing cantilevers and the formation of tips of various types and configurations. The cantilever arms have been made of fine tungsten wires with tips such as tiny diamond fragments, or other appropriate composition, secured thereon. Also, cantilevers have been fabricated using photo lithographic techniques, but such techniques did not produce satisfactory tips. Etching of silicon wafers has been used to produce the cantilevers, and more recently processes used in the silicon semiconductor integrated circuit industry have been used. These latter prior art cantilever fabrication processes are exemplified by U.S. Pat. Nos. 4,943,719 dated Jul. 24, 1990 and No. 5,021,364 dated Jun. 4, 1991, each issued to S. Akamine et al.
While these prior fabrication techniques and AFM apparatus have been successfully employed to image biological molecules with nanometer resolution, and micro-manipulators have been designed to isolate and hold single cells, but these prior approaches do not have the capability to cut and to move parts of biological molecules to predetermined locations. Flow sorting can be used to isolate cells and chromosomes tagged with fluorochromes and to direct them onto slides or into test tubes, but it cannot discriminate between and isolate structures containing subtle morphological differences. Thus, there is a need for a technique and apparatus to image small objects (such as human chromosomes, with nanometer resolution and to dissect cells or chromosomes and to isolate and collect nanometer-size fragments or organelles.
The present invention satisfies this need by providing instrumentation and techniques to image such small objects, to cut off identified parts of such objects, and to move or manipulate such cut-off parts on a substrate on which they are being imaged to predetermined locations on the substrate for collecting the desired cut-off parts. Also, the invention provides a capability for measuring hardness of small objects.